Isothiazolinone Biocides Enhanced By Zinc Ions

ABSTRACT

The present invention relates to an antimicrobial composition comprising an isothiazolinone, such as 1,2-benzisothiazolin-3-one, and a zinc compound selected from zinc salts, zinc oxides, zinc hydroxides or combinations thereof. Useful zinc salts include for example, oxides, sulfates, chlorides, and combinations thereof. In use, the zinc from the zinc compound enhances the antimicrobial activity to the isothiazolin-containing composition. This enhancement permits achieving the desired antimicrobial activity at a lower usage rate than is achieved using the isothiazolinone in the absence of the zinc compound. The antimicrobial composition can also contain co-biocides, such as pyrithiones, including zinc pyrithione or copper pyrithione.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/808,697, filed May 26, 2006, the disclosure of which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Isothiazolinones, such as 1,2-benzisothiazolin-3-one (also referred toas “BIT”), are know to be effective antimicrobials. BIT, as disclosedfor example in formulations described in European patent EP 0 703 726,is widely used as a bactericide in a variety of applications.Illustratively, British patent publication GB2,230,190A discloses apreservative composition containing BIT and an adduct of zinc chloride(“ZC”), together with 2,2′-dithiopyridine-1,1′-dioxide (“DTP”). Example3 of '190A publication compares compositions containing BIT plus ZC plusDTP against a control example containing only BIT plus ZC. The resultsgiven in Example 3 and Table 3 of the publication indicate thatinclusion of DTP allows decreased usage of BIT in the composition.Nonetheless, the amount of BIT (2.5 ppm) employed in the control examplehas been found by the present inventors to be insufficient forantimicrobial efficacy in real-world applications.

While BIT has proven useful in a wide range of applications, the usefulamount of BIT that can be added to a commercial product is limited byefficacy and economic considerations and to a lesser extent byenvironmental and toxicological concerns. Accordingly, alternativeantimicrobial compositions are needed for wet state applications thatare cost-effective and minimize the likelihood of adverse environmentaland toxicological effects. The present invention provides one suchalternative.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an antimicrobialcomposition comprising (a) at least one isothiazolin-3-one and (b) atleast one zinc compound selected from the group consisting of zincsalts, zinc oxides, zinc hydroxides, and combinations thereof.Illustrative zinc salts are zinc chloride, zinc sulfide, zinc carbonate,basic zinc carbonate, and combinations thereof. The zinc compoundprovides a source of metal ion in the antimicrobial composition. In thecomposition, the isothiazolin-3-one is present in an amount of from 1 to500 ppm (preferably from 5 to 500 ppm), the zinc compound is present inan amount from 5 to 200,000 ppm (preferably from 5 to 500 ppm), and theweight ratio of the isothiazolin-3-one to the zinc compound is from1:2000 to 100:1 (preferably from 1:100 to 100:1). Optionally, theantimicrobial composition additionally comprises a pyrithione salt orpyrithione acid. Useful pyrithione salts include copper pyrithione, zincpyrithione, sodium pyrithione, pyrithione disulfide, and combinationsthereof.

In another aspect, the present invention relates to an antimicrobialcomposition concentrate that, upon dilution with water, providesantimicrobial efficacy in a functional fluid. The concentrate comprises(a) at least one isothiazolin-3-one and (b) at least one zinc compoundselected from the group consisting of zinc salts, zinc oxides, zinchydroxides, and combinations thereof. In the concentrate, component (a)is present in an amount of from 1 to 95% w/w, and component (b) ispresent in an amount of from 1 to 50% w/w based on the total weight ofthe concentrate, with the proviso that the weight percent of (a) plus(b) does not exceed 100% by weight.

In yet another aspect, the present invention relates to functional fluidthat comprises a base medium plus (a) at least one isothiazolin-3-oneand (b) at least one zinc compound selected from the group consisting ofzinc salts, zinc oxides, zinc hydroxides, and combinations thereof.Illustrative zinc salts are zinc chloride, zinc sulfide, zinc carbonate,basic zinc carbonate, and combinations thereof. In the composition, theisothiazolin-3-one is present in an amount of from 1 to 500 ppm(preferably from 5 to 500 ppm), the metal compound is present in anamount of from 5 to 200,00 ppm (preferably from 5 to 500 ppm), and theweight ratio of the isothiazolin-3-one to zinc ion is from 1:2000 to100:1 (preferably from 100:1 to 1:100). Optionally, the antimicrobialcomposition additionally comprises a pyrithione salt or pyrithione acid.Useful pyrithione salts include copper pyrithione, zinc pyrithione,sodium pyrithione, and pyrithione disulfide. The base medium for thefunctional fluid can be, for example, a polymer useful in polymeremulsions. Examples of polymer systems are lattices, such as acrylic andsubstituted (meth)acrylates, styrene/butadiene, ethylene vinyl acetate,polyvinyl acetate, styrene/butadiene/N-methylol acrylamide, nitrile andcopolymers of the aforementioned. Typical functional fluids includecoating compositions, such as paints, adhesives, sealants, caulks,mineral and pigment slurries, printing inks, agricultural pesticideformulations, household products, personal care, metal working fluidsand other aqueous based systems.

These and other aspects will become apparent upon reading the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a graphical depiction of an Isobologram showingAntimicrobial Activity of Mixtures of 1,2-Benzisothiazolin-3-one (“BIT”)and Zinc Chloride against Ps. aeruginosa based on Fractional InhibitoryConcentration values.

FIG. 2 provides a graphical depiction of an Isobologram showingAntimicrobial Activity of Mixtures of Kathon® (a mixture of5-chloro-2-methyl-4-isothiazolin-3-one (“CIMIT”) and2-methyl-4-isothiazolin-3-one (“MIT”)) and Zinc Chloride against Ps.aeruginosa based on Fractional Inhibitory Concentration values.

FIG. 3 provides a graphical depiction of an Isobologram showingAntimicrobial Activity of Mixtures of MIT and Zinc Chloride against Ps.aeruginosa based on Fractional Inhibitory Concentration values.

FIG. 4 provides a graphical depiction of an Isobologram showingAntimicrobial Activity of Mixtures ofN-(n-butyl)-1,2-benzisothiazolin-3-one (“BBIT”) and Zinc Chlorideagainst Ps. aeruginosa based on Fractional Inhibitory Concentrationvalues.

FIG. 5 provides a graphical depiction of an Isobologram showingAntimicrobial Activity of Mixtures of BIT and Sodium Chloride againstPs. aeruginosa based on Fractional Inhibitory Concentration values.

DETAILED DESCRIPTION OF THE INVENTION

It has now been surprisingly found in accordance with the presentinvention that an antimicrobial composition containing anisothiazolin-3-one, plus a zinc compound selected from the groupconsisting of zinc salts, zinc oxides, zinc hydroxides, and combinationsthereof, exhibits enhanced antimicrobial efficacy, as compared toisothiazolin-containing compositions that do not contain the metalcompound. Enhanced efficacy are suitably provided in antimicrobialcompositions over a wide range of pHs of from 3 to 12.

The antimicrobial composition of the present invention is suitablyincorporated into a functional fluid. The functional fluid suitablycomprises a base medium plus (a) at least one isothiazolin-3-one and (b)at least one zinc compound selected from the group consisting of zincsalts of organic acids, zinc salts of inorganic acids, zinc oxides, zinchydroxides, and combinations thereof. Illustrative zinc salts includezinc chloride, zinc sulfide, zinc carbonate, basic zinc carbonate (alsoknown as “hydroxy-containing zinc carbonate”, also know as “zinc hydroxycarbonate” which is further identified by the empirical formulaZn₅(OH)₆(CO₃)₂), and combinations thereof.

The isothiazolinone useful in the present invention is preferably anisothiazolin-3-one that is selected from: 1,2-benzisothiazolin-3-one(“BIT”), N-(n-butyl)-1,2-benzisothiazolin-3-one (“BBIT”),4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (“DCOIT”),2-methyl-4-isothiazolin-3-one (“MIT”), mixtures of5-chloro-2-methyl-4-isothiazolin-3-one (“CIMIT”) plus MIT (availablefrom Rohm and Haas Company, Philadelphia, Pa. under the tradenameKathon®) dithio-2,2′-bis(benzmethylamide), and combinations thereof.Particularly preferred isothiazolins are BIT, MIT and BBIT, andcombinations thereof.

In the composition, the isothiazolin-3-one is present in an amount offrom 1 to 500 ppm (preferably from 5 to 500 ppm), the zinc compound ispresent in an amount of from 5 to 200,000 ppm (preferably from 5 to 500ppm), and the weight ratio of the isothiazolin-3-one to the zinccompound is from 1:2000 to 100:1 (preferably from 100:1 to 1:100).

Optionally, the antimicrobial composition additionally comprises apyrithione salt or pyrithione acid. Useful pyrithione salts includecopper pyrithione, zinc pyrithione, sodium pyrithione. If used, thepyrithione salt or pyrithione acid is suitably present in thecomposition in an amount of from about 0.001% w/w to about 1.0% w/w.

In one embodiment, the combination of antimicrobial components for theantimicrobial composition can be provided in the form of anantimicrobial composition concentrate that, upon dilution with water,provides antimicrobial efficacy in a functional fluid. The concentratecomprises (a) at least one isothiazolin-3-one and (b) at least one zinccompound selected from the group consisting of zinc salts, zinc oxides,zinc hydroxides, and combinations thereof. In the concentrate, component(a) is present in an amount of from 1 to 95% w/w, and component (b) ispresent in an amount of from 1 to 50% w/w.

The antimicrobial compositions of the present invention are suitablyused in functional fluids such as polymer emulsions, or other coatingcompositions, to impart both wet state and dry film preservation. Thebase medium can be, for example, a polymer useful in polymer emulsionswherein the polymer is selected from the group consisting of acrylic andsubstituted (meth)acrylates, styrene/butadiene, ethylene vinyl acetate,polyvinyl acetate, styrene/butadiene/N-methylol acrylamide, nitrile andcopolymers of the aforementioned. Typical functional fluids includecoating compositions, such as paints, adhesives, sealants, caulks,mineral and pigment slurries, printing inks, agricultural pesticideformulations, household products, personal care, metal working fluidsand other aqueous based systems.

The invention is further described in the Examples given below. Allpercentages given herein are weight percents based on the total weightof the composition, unless otherwise stated. All patents referred to inthis application are incorporated herein by reference in their entirety.

Example 1

In this example, samples of a commercial water based acrylic latex,trade name REVACRYL 1A, supplied by the Harlow Chemical Company, pH 8.1,containing antimicrobial additives in amounts as identified in Table Ibelow, were challenged with a bacterial suspension consisting of

Pseudomonas aeruginosa, NCIMB 8295Providencia rettgeri, NCIMB 10842Serratia marcescens, NCIMB 9523Aeromonas hydrophila, NCIMB9233Alcaligenes spp. (Wet State Preservation isolate)Burkholderia cepacia, ATCC 25416Pseudomonas putida, NCIMB9494

All test species were cultured onto nutrient agar and incubated for 24hours at 30° C. After this period individual suspensions of each testorganism were prepared at a concentration of 10⁶ colony forming unitsper ml in saline solution by means of a Thoma counting chamber, achievedby mixing equal volumes of the individual suspensions. The concentrationof bacteria in each aliquot after each challenge was 2×10⁶ per ml.

The antimicrobial additive-containing paint samples were stored at 40°C. for 7 days prior to commencing the test. The paint samples werechallenged with 1% v/v of bacterial suspension as described above. Alltest samples were incubated at 30° C. for the duration of the test andexamined for viable bacteria after 1, 3 and 7 days after each challenge.Viable bacteria were detected by streaking aliquots onto nutrient agarfollowed by incubation at 30° C. for 2 days.

Table I presents the results obtained after the third inoculation days1, 3 and 7.

TABLE I 3^(rd) inoculation Sample BIT/ppm Day 1 Day 3 Day 7 BIT/ZnO 50 CC C 100 1+ (92) 1+ (27) − 150 1+ (95) S (7) − 200  S (10) T (4) − 250 1+(89) 1+ (22) − 300 1+ (77) 1+ (12) − BIT/ZnCl2 50 C C C 100 ++ 1+ (30) −150 1+ (34) 1+ (21) − 200 1+ (51) 1+ (14) − 250  ++ (185)  l+ (25) − 3001+ (58) 1+ (17) − PROXEL BD20 50 C C C 100 C C C 150 C C C 200 C C C 2501+ (75) − − 300  l+ (54) − − 400 1+ (47) − − Assessments are rated as: −no growth detected T 0-5 colonies present S 5-10 colonies 1+ 20-100colonies + light growth - no specified number of colonies ++ moderategrowth - colonies visible, some coalescence +++ heavy growth -coalescing colonies visible throughout C confluent heavy growth

Results demonstrate a potentiating effect of BIT in combination with azinc containing salt when compared against a commercial product, namelyPROXEL BD20 a product of Arch Chemicals, Inc. containing only BIT forthe preservation of a commercial latex in that only 100 ppm of BIT isrequired in the presence of a zinc containing salt compared to 250 ppmof BIT for sample containing only BIT.

Example 2

Investigation of Potentiation Between 1,2-Benzisothiazolin-3-One (BIT)and Zinc Chloride (ZnCl₂) Against Pseudomonas aeruginosa.

Bacteria

Pseudomonas aeruginosa (NCIB 10421) maintained on nutrient agar, wereused to study the synergy between BIT and ZnCl₂.

Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures.

Bacteria were grown to stationary phase (approximately 24 hours) innutrient broth (approximately 10⁹ organisms per ml). A 0.1% (v/v)inoculum was used to seed fresh medium and 100 μl of the inoculum wasthen added to each well of a microtitre plate, except for the first wellwhich contained 200 μl.

Using doubling dilutions, the concentration of the compounds underinvestigation were varied in each well along the ordinate axis. Thepresence or absence of growth was recorded by visual inspection after 24hours incubation at 37° C.

Calculation of Antimicrobial Activity Against Mono-Cultures

Microtitre plates were used for this assay. A simple matrix wasconstructed with varied concentrations of the two compounds from 2×MIC(minimum inhibitory concentration) down to zero concentration in a 10×10array. As the microtitre plate has only 96 wells, the combinations ofthe two compounds that made up the extreme concentrations (highest andlowest) were omitted. Solutions were made up in broth at two times thefinal concentrations after pre-dissolving the compounds in distilledwater.

The mixture (100 μl) was added to the plate so that the total volume ineach well was 200 μl. Nutrient broth was used for Ps. aeruginosa. Plateswere incubated for 16-24 hours at 37°. The presence or absence of growthwas determined by visual inspection.

Results for Example 2

TABLE 2 Table 2: Antimicrobial Activity of Compounds Under InvestigationMIC (ppm) against Ps. aeruginosa COMPOUND 1 2 3 4 BIT 30 42 42 30 ZnCl₂300 450 375 450 BIT is 1,2-benzisothiazolin-3-one available from ArchLimited PROXEL ® GXL.

Calculation of Activity Against Mono-Cultures

The Minimum Inhibitory Concentration (MIC) is the lowest concentrationof biocide which showed growth inhibition when used alone. For thepurpose of Fractional Inhibitory Concentration (FIC) calculations, if asingle biocide did not control growth, the MIC was taken as the highestconcentration used. A Fractional Inhibitory Concentration is theconcentration of biocide which controlled growth in the mixture dividedby the MIC of that biocide. FIC values for both compounds in the mixturewere calculated and the results are shown in Table 3. The sum of thesetwo figures gives an indication of the action of the two biocides. Avalue less than one indicates an enhanced effect, if the total is unityor greater the action is additive and if the value is greater than twothe biocides are antagonistic.

TABLE 3 Fractional Inhibitory Concentrations for1,2-Benzisothiazolin-3-one (BIT) and Zinc Chloride (ZnCl₂) against Ps.aeruginosa FIC VALUES COMPOUND 1 2 3 4 BIT 1 0.20 0.20 0.40 0.14 0.140.29 0.29 0.43 0.14 0.20 0.40 0 ZnCl₂ 0 0.25 0.50 0.25 0.33 0.50 0.170.33 0.17 0.60 0.33 0.17 1 Total 1 0.45 0.70 0.65 0.47 0.64 0.46 0.620.60 0.74 0.53 0.57 1

If a graph with the axes representing the biocide Fractional InhibitoryConcentrations for the two biocides on linear scales is constructed,when the combination is additive the isobole (i.e. the line joining thepoints that represent all combinations with the same effect includingthe equally effective concentrations of the biocides used alone) isstraight, enhanced combinations give concave isoboles and antagonisticcombinations give convex isoboles. As shown in FIG. 1, the combinationof BIT and ZnCl₂ has an enhanced effect against Ps. aeruginosa asconfirmed by the concave isobole on the graph of FIG. 1.

Example 3

Investigation of potentiation between Kathon® (CIMIT/MIT) and ZincChloride (ZnCl₂) against Pseudomonas aeruginosa.

Bacteria

Pseudomonas aeruginosa (NCIB 10421) maintained on nutrient agar, wereused to study the synergy of Kathon® and ZnCl₂.

Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures

Bacteria were grown to stationary phase (approximately 24 hours) innutrient broth (approximately 10⁹ organisms per ml). A 0.1% (v/v)inoculum was used to seed fresh medium and 100 μl of the inoculum wasthen added to each well of a microtitre plate, except for the first wellwhich contained 200 μl.

Using doubling dilutions, the concentration of the compounds underinvestigation were varied in each well along the ordinate axis. Thepresence or absence of growth was recorded by visual inspection after 24hours incubation at 37° C.

Calculation of Antimicrobial Activity Against Mono-Cultures

Microtitre plates were used for this assay. A simple matrix wasconstructed with varied concentrations of the two compounds from 2×MIC(minimum inhibitory concentration) down to zero concentration in a 10×10array. As the microtitre plate has only 96 wells, the combinations ofthe two compounds that made up the extreme concentrations (highest andlowest) were omitted. Solutions were made up in broth at two times thefinal concentrations after pre-dissolving the compounds in distilledwater.

The mixture (100 μl) was added to the plate so that the total volume ineach well was 200 μl. Nutrient broth was used for Ps. aeruginosa. Plateswere incubated for 16-24 hours at 37°. The presence or absence of growthwas determined by visual inspection.

Results of Example 3

TABLE 4 Table 4: Antimicrobial Activity of Compounds Under InvestigationMIC (ppm) against Ps. aeruginosa COMPOUND 1 2 3 4 Kathon 0.9 0.9 0.90.53 ZnCl₂ 294 294 210 600

The mixture of CIMIT and MIT is available from Rohm and Haas Company,Philadelphia, Pa. under the tradename Kathon®.

Calculation of Synergy Against Mono-Cultures

The Minimum Inhibitory Concentration (MIC) is the lowest concentrationof biocide which showed growth inhibition when used alone. For thepurpose of Fractional Inhibitory Concentration (FIC) calculations, if asingle biocide did not control growth, the MIC was taken as the highestconcentration used. A Fractional Inhibitory Concentration is theconcentration of biocide which controlled growth in the mixture dividedby the MIC of that biocide. FIC values for both compounds in the mixturewere calculated and the results are shown in Table 5. The sum of thesetwo figures gives an indication of the action of the two biocides. Avalue less than one indicates an enhanced effect, if the total is unityor greater the action is additive and if the value is greater than twothe biocides are antagonistic.

TABLE 5 Fractional Inhibitory Concentrations for Kathon (CIMIT/MIT) andZinc Chloride (ZnCl₂) against Ps. aeruginosa FIC VALUES COMPOUND 1 2 3 4Kathon 1 0.17 0.17 0.33 0.33 0.17 0.33 0.33 0.14 0.28 0.28 0 ZnCl₂ 00.14 0.29 0.14 0.29 0.29 0.14 0.29 0.50 0.38 0.50 1 Total 1 0.31 0.460.47 0.62 0.46 0.47 0.62 0.64 0.66 0.78 1

If a graph with the axes representing the biocide Fractional InhibitoryConcentrations for the two biocides on linear scales is constructed,when the combination is additive the isobole (i.e. the line joining thepoints that represent all combinations with the same effect includingthe equally effective concentrations of the biocides used alone) isstraight, enhanced combinations give concave isoboles and antagonisticcombinations give convex isoboles. As shown in FIG. 2, the combinationof Kathon® and ZnCl₂ has an enhanced effect against Ps. aeruginosa asconfirmed by the concave isobole in FIG. 2.

Example 4

Investigation of Potentiation Between MIT and Zinc Chloride (ZnCl₂)Against Pseudomonas aeruginosa

Bacteria

Pseudomonas aeruginosa (NCIB 10421) maintained on nutrient agar, wereused to study the synergy between MIT and ZnCl₂.

Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures

Bacteria were grown to stationary phase (approximately 24 hours) innutrient broth (approximately 10⁹ organisms per ml). A 0.1% (v/v)inoculum was used to seed fresh medium and 100 μl of the inoculum wasthen added to each well of a microtitre plate, except for the first wellwhich contained 200 μl.

Using doubling dilutions, the concentration of the compounds underinvestigation were varied in each well along the ordinate axis. Thepresence or absence of growth was recorded by visual inspection after 24hours incubation at 37° C.

Calculation of Antimicrobial Activity Against Mono-Cultures

Microtitre plates were used for this assay. A simple matrix wasconstructed with varied concentrations of the two compounds from 2×MIC(minimum inhibitory concentration) down to zero concentration in a 10×10array. As the microtitre plate has only 96 wells, the combinations ofthe two compounds that made up the extreme concentrations (highest andlowest) were omitted. Solutions were made up in broth at two times thefinal concentrations after pre-dissolving the compounds in distilledwater.

The mixture (100 μl) was added to the plate so that the total volume ineach well was 200 μl. Nutrient broth was used for Ps. aeruginosa. Plateswere incubated for 16-24 hours at 37°. The presence or absence of growthwas determined by visual inspection.

Results of Example 4

TABLE 6 Table 6: Antimicrobial Activity of Compounds Under InvestigationMIC (ppm) against Ps. aeruginosa COMPOUND 1 2 3 4 MIT 9 6 10.5 8 ZnCl₂210 210 450 450

Calculation of Synergy Against Mono-Cultures

The Minimum Inhibitory Concentration (MIC) is the lowest concentrationof biocide which showed growth inhibition when used alone. For thepurpose of Fractional Inhibitory Concentration (FIC) calculations, if asingle biocide did not control growth, the MIC was taken as the highestconcentration used. A Fractional Inhibitory Concentration is theconcentration of biocide which controlled growth in the mixture dividedby the MIC of that biocide. FIC values for both compounds in the mixturewere calculated and the results are shown in Table 7. The sum of thesetwo figures gives an indication of the action of the two biocides. Avalue less than one indicates an enhanced effect, if the total is unityor greater the action is additive and if the value is greater than twothe biocides are antagonistic.

TABLE 7 Fractional Inhibitory Concentrations for (MIT) and Zinc Chloride(ZnCl₂) against Ps. aeruginosa FIC Values COMPOUND 1 2 3 4 MIT 1 0.170.33 0.33 0.50 0.25 0.50 0.75 0.14 0.29 0.17 0 ZnCl₂ 0 0.57 0.29 0.430.14 0.43 0.29 0.14 0.33 0.17 0.50 1 Total 1 0.74 0.62 0.76 0.64 0.680.79 0.89 0.48 0.46 0.67 1

If a graph with the axes representing the biocide Fractional InhibitoryConcentrations for the two biocides on linear scales is constructed,when the combination is additive the isobole (i.e. the line joining thepoints that represent all combinations with the same effect includingthe equally effective concentrations of the biocides used alone) isstraight, enhanced combinations give concave isoboles and antagonisticcombinations give convex isoboles. As shown in FIG. 3, the combinationof MIT and ZnCl₂ has an enhanced effect against Ps. aeruginosa confirmedby the concave isobole in FIG. 3.

Example 5

Investigation of Potentiation Between BBIT and Zinc Chloride (ZnCl₂)Against Pseudomonas aeruginosa.

Bacteria

Pseudomonas aeruginosa (NCIB 10421) maintained on nutrient agar, wereused to study the synergy between BBIT and ZnCl₂.

Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures

Bacteria were grown to stationary phase (approximately 24 hours) innutrient broth (approximately 10⁹ organisms per ml). A 0.1% (v/v)inoculum was used to seed fresh medium and 100 μl of the inoculum wasthen added to each well of a microtitre plate, except for the first wellwhich contained 200 μl.

Using doubling dilutions, the concentration of the compounds underinvestigation were varied in each well along the ordinate axis. Thepresence or absence of growth was recorded by visual inspection after 24hours incubation at 37° C.

Calculation of Antimicrobial Activity Against Mono-Cultures

Microtitre plates were used for this assay. A simple matrix wasconstructed with varied concentrations of the two compounds from 2×MIC(minimum inhibitory concentration) down to zero concentration in a 10×10array. As the microtitre plate has only 96 wells, the combinations ofthe two compounds that made up the extreme concentrations (highest andlowest) were omitted. Solutions were made up in broth at two times thefinal concentrations after pre-dissolving the compounds in distilledwater.

The mixture (100 μl) was added to the plate so that the total volume ineach well was 200 μl. Nutrient broth was used for Ps. aeruginosa. Plateswere incubated for 16-24 hours at 37°. The presence or absence of growthwas determined by visual inspection.

Results of Example 5

TABLE 8 Table 8: Antimicrobial Activity of Compounds Under InvestigationMIC (ppm) against Ps. aeruginosa COMPOUND 1 2 3 4 BBIT 315 360 42 180ZnCl₂ 525 525 600 600 BBIT is N-(n-butyl)-1,2-benzisothiazolin-3-one.

BBIT is N-(n-butyl)-1,2-benzisothiazolin-3-one.

Calculation of Synergy Against Mono-Cultures

The Minimum Inhibitory Concentration (MIC) is the lowest concentrationof biocide which showed growth inhibition when used alone. For thepurpose of Fractional Inhibitory Concentration (FIC) calculations, if asingle biocide did not control growth, the MIC was taken as the highestconcentration used. A Fractional Inhibitory Concentration is theconcentration of biocide which controlled growth in the mixture dividedby the MIC of that biocide. FIC values for both compounds in the mixturewere calculated and the results are shown in Table 9. The sum of thesetwo figures gives an indication of the action of the two biocides. Avalue less than one indicates an enhanced effect, if the total is unityor greater the action is additive and if the value is greater than twothe biocides are antagonistic.

TABLE 9 Fractional Inhibitory Concentrations for BBIT and Zinc Chloride(ZnCl₂) against Ps. aeruginosa FIC values 1 2 3 4 BBIT 1 0.29 0.25 0.140.71 0.25 1 ZnCl₂ 0 0.43 0.43 0.50 0.38 0.38 1 Total 1 0.71 0.68 0.641.09 0.63 1

If a graph with the axes representing the biocide Fractional InhibitoryConcentrations for the two biocides on linear scales is constructed,when the combination is additive the isobole (i.e. the line joining thepoints that represent all combinations with the same effect includingthe equally effective concentrations of the biocides used alone) isstraight, enhanced combinations give concave isoboles and antagonisticcombinations give convex isoboles. As shown in FIG. 4, the combinationof BBIT and ZnCl₂ has an enhanced effect against Ps. aeruginosa asconfirmed by the concave isobole shown in FIG. 4.

Comparative Example A

Investigation of Potentiation Between 1,2-Benzisothiazolin-3-One (BIT)and Sodium Chloride (NaCl₂) Against Pseudomonas aeruginosa as Comparedto the Synergy Between 1,2-Benzisothiazolin-3-One (BIT) and ZincChloride (ZnCl₂) Against Pseudomonas aeruginosa (Example 2)

Bacteria

Pseudomonas aeruginosa (NCIB 10421) maintained on nutrient agar, wereused to study the efficacy of the combination of BIT with sodiumchloride.

Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures

Bacteria were grown to stationary phase (approximately 24 hours) innutrient broth (approximately 10⁹ organisms per ml). A 0.1% (v/v)inoculum was used to seed fresh medium and 100 μl of the inoculum wasthen added to each well of a microtitre plate, except for the first wellwhich contained 200 μl.

Using doubling dilutions, the concentration of the compounds underinvestigation were varied in each well along the ordinate axis. Thepresence or absence of growth was recorded by visual inspection after 24hours incubation at 37° C.

Calculation of Antimicrobial Activity Against Mono-Cultures

Microtitre plates were used for this assay. A simple matrix wasconstructed with varied concentrations of the two compounds from 2×MIC(minimum inhibitory concentration) down to zero concentration in a 10×10array. As the microtitre plate has only 96 wells, the combinations ofthe two compounds that made up the extreme concentrations (highest andlowest) were omitted. Solutions were made up in broth at two times thefinal concentrations after pre-dissolving the compounds in distilledwater.

The mixture (100 μl) was added to the plate so that the total volume ineach well was 200 μl. Nutrient broth was used for Ps. aeruginosa. Plateswere incubated for 16-24 hours at 37°. The presence or absence of growthwas determined by visual inspection.

Results of Comparative Example A

TABLE 10 Table 10: Antimicrobial Activity of Compounds UnderInvestigation COMPOUND MIC (ppm) against Ps. aeruginosa BIT 30 NaCl₂None detected (greater than 675) BIT is 1,2-benzisothiazolin-3-oneavailable from Arch Limited as PROXEL^(RTM) GXL.

Calculation of Potentiation Against Mono-Cultures

The Minimum Inhibitory Concentration (MIC) is the lowest concentrationof biocide which showed growth inhibition when used alone. For thepurpose of Fractional Inhibitory Concentration (FIC) calculations, if asingle biocide did not control growth, the MIC was taken as the highestconcentration used. A Fractional Inhibitory Concentration is theconcentration of biocide which controlled growth in the mixture dividedby the MIC of that biocide. FIC values for both compounds in the mixturewere calculated and the results are shown in Table 11. The sum of thesetwo figures gives an indication of the action of the two biocides. Avalue less than one indicates an enhanced effect, if the total is unityor greater the action is additive and if the value is greater than twothe biocides are antagonistic.

TABLE 11 Table 11: Fractional Inhibitory Concentrations for1,2-Benzisothiazolin-3-one (BIT) and Sodium Chloride (NaCl₂) against Ps.aeruginosa COMPOUND FIC VALUES BIT 1 1.00 0 NaCl₂ 0 0.33 1 Total 1 1.331

If a graph with the axes representing the biocide Fractional InhibitoryConcentrations for the two biocides on linear scales is constructed,when the combination is additive the isobole (i.e. the line joining thepoints that represent all combinations with the same effect includingthe equally effective concentrations of the biocides used alone) isstraight, enhanced combinations give concave isoboles and antagonisticcombinations give convex isoboles. As shown in FIG. 5, the combinationof BIT and NaCl₂ is additive against Ps. aeruginosa as confirmed by thestraight isobole in FIG. 5. Additionally, the results of this Examplefurther illustrate that the combination of an isothiazolin-3-one such asBIT and a zinc compound such as ZnCl₂, as shown in Example 2, showunexpected potentiation results against Ps. aeruginosa.

What is claimed is:
 1. An antimicrobial composition comprising (a) atleast one isothiazolin-3-one and (b) at least one zinc compound selectedfrom the group consisting of zinc salts, zinc oxides, zinc hydroxides,and combinations thereof, wherein said isothiazolin-3-one is present inan amount of from 1 to 500 ppm, the zinc compound is present in anamount of from 5 to 200,000 ppm, and the weight ratio of theisothiazolin-3-one to the zinc compound is from 1:2000 to 100:1.
 2. Thecomposition of claim 1 wherein component (a) is present in an amount offrom 1 to 500 ppm, component (b) is present in an amount of from 5 to200,000 ppm, and the weight ratio is from 1:100 to 100:1.
 3. Thecomposition of claim 1 wherein component (a) is present in an amount offrom 5 to 500 ppm, and component (b) is present in an amount of from 5to 500 ppm.
 4. The antimicrobial composition of claim 1 wherein saidisothiazolin-3-one is selected from the group consisting of1,2-benzisothiazolin-3-one, N-(n-butyl)-1,2-benzisothiazolin-3-one,4,5-dichloro-2-n-octyl-4-isothiazolin-3-one,2-methyl-4-isothiazolin-3-one, mixtures of5-chloro-2-methyl-4-isothiazolin-3-one (CIMIT) and2-methyl-4-isothiazolin-3-one, and alsodithio-2,2′-bis(benzmethylamide), and combinations thereof.
 5. Theantimicrobial composition of claim 1 wherein said zinc salt is selectedfrom the group consisting of zinc chloride, zinc sulfide, zinccarbonate, basic zinc carbonate, and combinations thereof.
 6. Theantimicrobial composition of claim 1 wherein said zinc compound is zincchloride.
 7. The antimicrobial composition of claim 1 which furthercomprises a co-biocide selected from the group consisting of pyrithionesalts, pyrithione acids, and combinations thereof, and wherein saidco-biocide is present in said composition in an amount of from about0.001 to about 1% w/w based on the total weight of said composition. 8.An antimicrobial composition concentrate, which upon dilution with waterprovides the amounts of components (a) and (b) as specified in claim 1,said concentrate comprising isothiazolin-3-one in an amount of between 1and 95% w/w.
 9. The antimicrobial composition concentrate of claim 8which further comprises a co-biocide selected from the group consistingof pyrithione salts, pyrithione acids, and combinations thereof, andwherein said co-biocide is present in said composition in an amount offrom about 1 to about 50% w/w based on the total weight of saidcomposition.
 10. A composition comprising a polymer emulsion, saidcomposition additionally comprising a wet state preservative comprisinga combination of (a) at least one isothiazolin-3-one and (b) at leastone zinc compound selected from the group consisting of zinc salts, zincoxides, zinc hydroxides, and combinations thereof, wherein saidcombination is present in said composition in a total amount of betweenabout 10 ppm and about 400 ppm, and wherein the molar ratio of component(a) to component (b) is between about 4:1 and about 1:2.
 11. Thecomposition of claim 10 wherein said polymer emulsion comprises apolymer selected from the group consisting of acrylic and substituted(meth)acrylates, styrene/butadiene, ethylene vinyl acetate, polyvinylacetate, styrene/butadiene/N-methylol acrylamide, nitrile and copolymersthereof.
 12. The antimicrobial composition of claim 10 wherein said zincsalt is selected from the group consisting of zinc chloride, zincsulfide, zinc carbonate, basic zinc carbonate, and combinations thereof.13. The antimicrobial composition of claim 12 wherein said zinc salt iszinc chloride.
 14. The composition of claim 10 wherein saidisothiazolin-3-one is selected from the group consisting of:1,2-benzisothiazolin-3-one, N-(n-butyl)-1,2-benzisothiazolin-3-one,4,5-dichloro-2-n-octyl-4-isothiazolin-3-one,2-methyl-4-isothiazolin-3-one, mixtures of5-chloro-2-methyl-4-isothiazolin-3-one (CIMIT) and2-methyl-4-isothiazolin-3-one, and alsodithio-2,2′-bis(benzmethylamide), and combinations thereof.
 15. Thecomposition of claim 10 which further comprises a co-biocide selectedfrom the group consisting of pyrithione salts, pyrithione acids, andcombinations thereof, and wherein said co-biocide is present in saidcomposition in an amount of from about 0.001 to about 1.0% w/w based onthe total weight of said composition.
 16. The composition of claim 15wherein said pyrithione salts are selected from the group consisting ofcopper pyrithione, zinc pyrithione, sodium pyrithione, and combinationsthereof.
 17. A coating composition selected from the group consisting ofpaints, adhesives, sealants, caulks, mineral and pigment slurries,printing inks, agricultural pesticide formulations, household coatingproducts, personal care products, metal working fluids, and combinationsthereof, wherein said coating composition contains a polymer emulsion asa liquid base medium, and a wet state preservative comprising acombination of (a) at least one isothiazolin-3-one and (b) at least onezinc compound selected from the group consisting of zinc salts, zincoxides, zinc hydroxides, and combinations thereof, wherein saidcombination is present in said composition in a total amount of betweenabout 10 ppm and about 400 ppm, and wherein the molar ratio of component(a) to component (b) is between about 4:1 and about 1:2.
 18. The coatingcomposition of claim 17 wherein said liquid base medium is selected fromthe group consisting of acrylic and substituted (meth)acrylates,styrene/butadiene, ethylene vinyl acetate, polyvinyl acetate,styrene/butadiene/N-methylol acrylamide, nitrile and copolymers thereof.